Neuropilin-1 aggravates liver cirrhosis by promoting angiogenesis via VEGFR2-dependent PI3K/Akt pathway in hepatic sinusoidal endothelial cells - PubMed (original) (raw)
Neuropilin-1 aggravates liver cirrhosis by promoting angiogenesis via VEGFR2-dependent PI3K/Akt pathway in hepatic sinusoidal endothelial cells
Le Wang et al. EBioMedicine. 2019 May.
Abstract
Background: We have revealed that neuropilin-1 (NRP-1) promoted hepatic stellate cell activation and liver fibrosis through its profibrogenic signalling pathways. However, the role of NRP-1 in angiogenesis in hepatic sinusoidal endothelial cells (HSECs) during liver cirrhosis remains unclear.
Methods: The correlation between NRP-1 expression and angiogenesis was evaluated in both human and murine cirrhotic liver tissues by immunohistochemical staining, quantitative real-time PCR, and western blotting. In addition, the role and mechanism of NRP-1 in regulating VEGFR2-dependent angiogenesis was identified in endothelial cells (ECs) in vitro. Moreover, liver histocultures were used to test the therapeutic effect of NRP-1 blocking in liver fibrosis.
Findings: Higher expression of NRP-1 in HSECs was detected, which was positively correlated with angiogenesis in liver cirrhosis. In vitro, NRP-1 knockdown suppressed the expression and activation of VEGFR2, accompanied by reduced ability of the vascular tube formation and the migration of ECs. Conversely, NRP-1 overexpression upregulated VEGFR2, promoted tube formation, and the migration of ECs. Mechanistically, NRP-1 modulated the expression of VEGFR2 by regulating FAK and its kinase activity. Furthermore, NRP-1 promoted VEGFR2-dependent angiogenesis via the PI3K/Akt pathway in HSECs. Blocking NRP-1 function reduced intrahepatic angiogenesis and fibrosis-associated factors in the in vitro liver histocultures.
Interpretation: NRP-1 promotes angiogenesis by upregulating the expression and activation of VEGFR2 through the PI3K/Akt signalling pathway in liver cirrhosis. This study highlights the possibility of therapeutically targeting NRP-1 for the treatment of cirrhosis. FUND: National Natural Science Foundation of China (No. 81570551; 81770607; 81600469; 81401868), Key Research project of Shandong Province (No. 2016GSF201008; 2017GSF218053), Natural Science Foundation of Shandong Province (No. ZR2017MH102), National Science and Technology Major Project of China (No. 2018ZX10302206-001-006).
Keywords: Cirrhosis; Hepatic sinusoidal endothelial cells; Intrahepatic angiogenesis; Neuropilin-1; Vascular growth factor receptor 2.
Copyright © 2019. Published by Elsevier B.V.
Figures
Fig. 1
NRP-1 upregulation in the HSECs is consistent with the enhancement of intrahepatic angiogenesis and fibrosis in human cirrhotic livers. (A) Representative histopathology images of human liver tissues with H&E staining (×50; scale bar = 200 μm), Masson staining (×50; scale bar = 200 μm), and immunohistochemistry images for NRP-1, VEGFR2, CD31, and α-SMA are shown for the normal group (up panels, n = 3) and the cirrhotic patients group (down panels, n = 7). NRP-1 and VEGFR2 were stained along the sinusoids, (×400; scale bar = 50 μm), CD31 in the intimal lining of the microvessels (×100; scale bar = 50 μm), and α-SMA in liver scar tissue appeared as brown deposits (×50; scale bar = 50 μm). (B) Western blotting showed that NRP-1, VEGFR2, CD31, and α-SMA protein levels in livers from cirrhotic patients were elevated compared with those in the normal group. (C) Both NRP-1 and VEGFR2 protein content were increased in human cirrhotic liver samples as assessed by western blotting (p = .0219 and p = .0067, respectively. **p < .01, *p < .05). (D) NRP-1 and VEGFR2 mRNA levels were analysed and compared in liver samples of cirrhotic patients and a control group. Both molecules were increased in human cirrhotic liver samples as assessed by qRT-PCR (p = .0193 and p = .0446, respectively. *p < .05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
NRP-1 expression is positively correlated with VEGFR2 expression and intrahepatic angiogenesis during cirrhosis progression in a cirrhotic mice model. (A) Time course expression of NRP-1 in control group mice livers (n = 4) and the CCl4-treated group over 6 or 8 weeks (n = 5). Liver tissues of mice treated with CCl4 were stained with H&E (×100; scale bar = 100 μm) and Masson (×50; scale bar = 200 μm) and immunostained using NRP-1(×400; scale bar = 50 μm), VEGFR2(×400; scale bar = 50 μm), CD31(×200; scale bar = 100 μm), and α-SMA ab (×100; scale bar = 500 μm). (B) Western blotting showed that NRP-1, VEGFR2, CD31, and α-SMA protein levels in CCl4-treated mice livers were increased in relation to the stage of cirrhosis. (C) NRP-1 and VEGFR2 mRNA levels were increased in accordance with the stage of cirrhosis of CCl4-treated mice liver samples by qRT-PCR (p = .0023 and p = .0147, respectively. **p < .01, *p < .05). (D) A significant correlation was observed between NRP-1 and VEGFR2 staining scores in mice liver samples (p = .0003, ***p < .001). (E) A significant correlation was observed between NRP-1 staining scores and CD31 staining in mice liver samples (p = .0009, ***p < .001).
Fig. 3
NRP-1 is required for HSECs capillary-like tube formation and cells migration in vitro. NRP-1 was downregulated or overexpressed with lentivirus in HSECs and HUVECs. (A) HSECs transfected with lentivirus NRP-1-RNAi showed diminished angiogenesis compared to cells transfected with the control via a tube formation assay and also showed impaired responses to VEGF. (B). Quantification of tubes in Matrigel was measured in NRP-1-RNAi and control groups in the presence of either vehicle or VEGF in HUVECs (n = 3, p = .038 and p = .0045, respectively. **p < .05, *p < .05). (C). Cell migration was also studied using Transwell assay in HSECs transfected with lentivirus NRP-1-RNAi or lentivirus control. Cells transduced with lentivirus NRP-1-RNAi showed decreased VEGF-dependent cell movement in comparison to cells transduced with lentivirus control. (D) The quantification of HUVECs that passed through the Transwell filter in different groups is shown (n = 3, p = .0112 and p = .0107, *p < .05). (E) HSECs transduced with lenti-NRP-1 showed enhanced tube formation compared with the lenti-control. (F) The quantification of HUVECs in tubes of Matrigel in the lenti-NRP-1 and lenti-control groups are shown (n = 3, p = .0317 and p = .0351, *p < .05). (G) Migration of HSECs in lenti-NRP-1 group increased in comparison with the lenti-control group. (H) The quantification of data in terms of number of HUVEC migrations was measured (n = 3, p = .0125 and p = .0272, *p < .05).
Fig. 4
NRP-1 upregulates VEGFR2 expression and activation via FAK and its activity. NRP-1 was downregulated or overexpressed with lentivirus in HSECs, HUVECs, and SK-HEP-1 cells. (A) NRP-1 mRNA was downregulated by NRP-1-RNAi compared with the control as confirmed by qRT-PCR (left, ***p < .001). Consistent with the decreased amount of NRP-1 mRNA, VEGFR2 mRNA expression was also decreased in ECs as confirmed by qRT-PCR (n = 3, ***p < .001). (B) NRP-1 mRNA was upregulated in lenti-NRP-1 group compared with lenti-control group as confirmed by qRT-PCR (left, p < .001, p < .001and p = .0037, respectively. ***p < .001, **p < .01). Consistent with the increased amount of NRP-1 mRNA, VEGFR2 mRNA expression also increased in ECs as confirmed by qRT-PCR (right) (n = 3, p = .0098, p = .0076 and p = .0331, respectively. **p < .01, *p < .05) (C) NRP-1 downregulation induced a significant reduction in the total level of VEGFR2 protein and phosphorylated VEGFR2 protein at Tyr1175 in ECs as confirmed by western blotting (left panel). NRP-1 overexpression enhanced the expression and phosphorylation of VEGFR2 at Tyr1175 as confirmed by western blotting (right panel). (D) Consistent with the changes in NRP-1 and VEGFR2 mRNA, the FAK mRNA level significantly decreased in HSECs transfected with NRP-1 RNAi compared with the control. In addition, FAK mRNA levels also increased in HSECs transfected with lenti-NRP-1 RNAi compared with the lenti-control (***p < .001). (E) NRP-1 overexpression enhanced the expression and phosphorylation of FAK at Y397 as seen in the western blot results. FAK inhibition blocks NRP-1–induced increases in the level of VEGFR2 expression and activation. (Representative WB are from three independent experiments).
Fig. 5
NRP-1 enhances VEGFR2 phosphorylation and downstream PI3K/Akt pathway activity. (A) HUVECs transduced with lenti-NRP-1 or lenti-control were stimulated with VEGF165 (10 ng/ml) for 0 and 15 min. NRP-1 overexpression enhanced the expression and phosphorylation of VEGFR2, as well as the phosphorylation of PI3K/Akt and PLCγ-1/ERK1/2 (representative WB are from 3 independent experiments). (B) PI3K/Akt and PLCγ-1/ERK1/2 activation levels increased in HSECs cells transduced with lenti-NRP-1 compared with HUVECs cells transduced with the lenti-control (n = 3, **p < .01, *p < .05). (C) Tube formation was analysed in HUVECs transduced with lenti-NRP-1 or the lenti-control. The PI3K inhibitor blocks NRP-1-induced increases in tube formation in the presence of either vehicle or VEGF (n = 3, **p < .01, *p < .05). (D) HSECs transduced with lenti-NRP-1 or the lenti-control were stimulated with VEGF165 (10 ng/ml) for 0 and 15 min. NRP-1 overexpression enhanced the expression and phosphorylation of VEGFR2, as well as the phosphorylation of PI3K/Akt. (Representative WB are from three independent experiments). (E) VEGFR2 and PI3K/Akt activation levels were increased in HSECs cells transduced with lenti-NRP-1 compared with HSECs cells transduced with the lenti-control (n = 3, **p < .01, *p < .05).
Fig. 6
Blocking NRP-1 function inhibits neoangiogenesis and fibrosis in vitro fibrotic liver tissue culture. (A) EG00229, a pharmacologic inhibitor of NRP-1 and sNRP-1, is a competitive soluble protein inhibitor of NRP-1 and was able to block enhanced angiogenesis conferred by lenti-NRP-1 overexpression in HSECs (n = 3, *p < .05). (B) Fibrotic liver tissues of patients administered vehicle (n = 4) or EG00229 (n = 4) in various drug concentrations (0,10 20,50 μM) were tested by CCK-8 after culturing for 7 days. The EG00229-treated fibrotic tissue showed an increased survival rate compared with vehicle-treated tissue, in relation to the drug concentration. (***p < .001) (C) Representative images for Masson staining in each liver tissue sample showed no significant differences between different treatment groups (original magnification, 40×). (D) Angiogenesis markers were quantified from the tissue sections in each group using qRT-PCR, with GAPDH as internal control. CD31 mRNA levels were significantly reduced in tissues treated with EG00229 compared with control tissues (p = .0019, **p < .01). (E–G) Fibrosis markers were also quantified using qRT-PCR, with GAPDH as internal control, from tissue sections pertaining to each group. Results showed that α-SMA, collagen1α I, and FN mRNA levels were significantly reduced in tissue treated with EG00229 compared with control tissues (p = .0002, p = .048, p = .0195, respectively. ***p < .001, *p < .05).
Fig. 7
Mechanistic role of NRP-1 in intrahepatic angiogenesis of liver cirrhosis. A proposed model depicting the role of NRP-1 in intrahepatic angiogenesis of liver cirrhosis. NRP-1 upregulates the expression and activation of VEGFR2 at transcriptional and post translational levels through upregulating FAK and its kinase activity. Furthermore, NRP-1 promotes intrahepatic angiogenesis by amplifying VEGFR2 phosphorylation and the downstream PI3K/Akt signalling pathway. Increases in NRP-1 correspond with liver cirrhosis progression and support the potential role of this molecule as a therapeutic target for liver cirrhosis treatment.
Supplementary Fig. 1
(A) Representative images of HSECs transfected by lentivirus NRP-1-RNAi and control confirmed by confocal microscope. (B) Representative images of HSECs transfected by lenti-NRP-1 and lenti-control confirmed by confocal microscope. (C) HUVECs transfected with lentivirus NRP-1-RNAi showed diminished proliferation compared to cells transfected with control in CCK-8 assay (n = 3, *p < .05). (D) HUVECs transduced with lenti-NRP-1 showed enhanced proliferation compared with the lenti-control in a time-dependent manner (n = 3, *p < .05). (E) HUVECs were stimulated with VEGF-A165 (10 ng/ml) for various time durations, from 0 to 60 min. Cell lysates were subjected to western blot assays with indicated Abs. VEGF-induced VEGFR2 and its downstream signals with peak phosphorylation at 15 min. (F) Tube formation assays were carried out in HUVECs transduced with lenti-NRP-1 or the lenti-control. The PI3K inhibitor blocks NRP-1–induced increases in tube formation in the presence of either vehicle or VEGF. (H) EG00229 and sNRP-1 blocked enhanced angiogenesis conferred by lenti-NRP-1 overexpression in HSEC in the presence of either vehicle or VEGF.
Supplementary Fig. 2
(A) NRP-1 overexpression enhanced the expression and phosphorylation of Akt at S473 as seen in western blot results. The PI3K inhibitor blocks NRP-1-induced increases in levels of Akt activation. (B) The NRP-1 protein was upregulated in the lenti-NRP-1 group compared with the lenti-control group, as confirmed by HSEC western blotting. (C–F) Results showed that angiogenesis and fibrosis markers were quantified using qRT-PCR with18S rRNA as internal control. CD31 mRNA levels were significantly reduced in tissues treated with EG00229 compared with control tissues (***p < .001). α-SMA, collagen1α I, and FN mRNA levels were significantly reduced in tissue treated with EG00229 compared with control tissues (p = .0043, p = .0118, p = .0103, respectively. **p < .001, *p < .05).
References
- Ferrara N., Gerber H.P., LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003;9(6):669–676. - PubMed
- He Z., Tessier-Lavigne M. Neuropilin is a receptor for the axonal chemorepellent Semaphorin III. Cell. 1997;90(4):739–751. - PubMed
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